Project Summary/Abstract Regulated proteolysis by the ubiquitin-proteasome system (ubiquitin system) plays essential roles in a multitude of biological processes and has major ramifications for human health and disease, including illnesses that range from cancer and neurodegeneration to cardiovascular syndromes and defects of immunity. Our studies of the ubiquitin-proteasome system and the ubiquitin-dependent N-end rule pathway over the last three decades were made possible, to a large extent, by the present grant (DK039520-30), currently in its 31st year of support. The N-end rule pathway recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins and thereby causes their degradation by the proteasome. Recognition components of the N-end rule pathway are called N-recognins. In eukaryotes, N-recognins are E3 ubiquitin ligases that can target N-degrons. The eukaryotic N-end rule pathway consists of three branches. The first branch, called the Arg/N-end rule pathway, targets specific unacetylated N-terminal residues of protein substrates. This branch, discovered by our laboratory in 1986, continues to be a fount of biological insights. The second branch, called the Ac/N-end rule pathway, was discovered by our laboratory in 2010. This pathway recognizes proteins that bear N-terminally acetylated residues. The third branch of the N-end rule pathway, termed the Pro/N-end rule pathway, has emerged through unpublished studies, over the last two years, by our laboratory. This proteolytic pathway is mediated by the previously known ubiquitin ligase called GID and recognizes the N-terminal proline (Pro) residue of a protein and an adjoining sequence motif. The present (DK039520) renewal application focuses on these (latest) advances and the largely unexplored Pro/N-end rule pathway, in both yeast and mammals. Its biological functions include the regulation of physiologically important transitions between cellular states that require de novo synthesis of glucose (for example, starvation or semi-starvation states) and conditions of ample glucose availability. The Pro/N-end rule pathway has other functions as well, which are beginning to emerge, in part through our recent identification of non-gluconeogenic proteins that are either targeted by or interact with the Pro/N-end rule pathway, in both yeast and mammals. These and related yeast and mammalian studies, described in Specific Aims of the present renewal application, will advance the understanding of protein degradation and the universally present N-end rule pathway.